Cytoskeletal Signaling: Is Memory Encoded in Microtubule Lattices by CaMKII Phosphorylation?

Memory is attributed to strengthened synaptic connections among particular brain neurons, yet synaptic membrane components are transient, whereas memories can endure. This suggests synaptic information is encoded and ‘hard-wired’ elsewhere, e.g. at molecular levels within the post-synaptic neuron. In long-term potentiation (LTP), a cellular and molecular model for memory, post-synaptic calcium ion (Ca2+) flux activates the hexagonal Ca2+-calmodulin dependent kinase II (CaMKII), a dodacameric holoenzyme containing 2 hexagonal sets of 6 kinase domains. Each kinase domain can either phosphorylate substrate proteins, or not (i.e. encoding one bit). Thus each set of extended CaMKII kinases can potentially encode synaptic Ca2+ information via phosphorylation as ordered arrays of binary ‘bits’. Candidate sites for CaMKII phosphorylation-encoded molecular memory include microtubules (MTs), cylindrical organelles whose surfaces represent a regular lattice with a pattern of hexagonal polymers of the protein tubulin. Using molecular mechanics modeling and electrostatic profiling, we find that spatial dimensions and geometry of the extended CaMKII kinase domains precisely match those of MT hexagonal lattices. This suggests sets of six CaMKII kinase domains phosphorylate hexagonal MT lattice neighborhoods collectively, e.g. conveying synaptic information as ordered arrays of six “bits”, and thus “bytes”, with 64 to 5,281 possible bit states per CaMKII-MT byte. Signaling and encoding in MTs and other cytoskeletal structures offer rapid, robust solid-state information processing which may reflect a general code for MT-based memory and information processing within neurons and other eukaryotic cells

Interaction of CK1δ with γTuSC ensures proper microtubule assembly and spindle positioning.

Casein kinase 1δ (CK1δ) family members associate with microtubule-organizing centers (MTOCs) from yeast to humans, but their mitotic roles and targets have yet to be identified. We show here that budding yeast CK1δ, Hrr25, is a γ-tubulin small complex (γTuSC) binding factor. Moreover, Hrr25's association with γTuSC depends on its kinase activity and its noncatalytic central domain. Loss of Hrr25 kinase activity resulted in assembly of unusually long cytoplasmic microtubules and defects in spindle positioning, consistent with roles in regulation of γTuSC-mediated microtubule nucleation and the Kar9 spindle-positioning pathway, respectively. Hrr25 directly phosphorylated γTuSC proteins in vivo and in vitro, and this phosphorylation promoted γTuSC integrity and activity. Because CK1δ and γTuSC are highly conserved and present at MTOCs in diverse eukaryotes, similar regulatory mechanisms are expected to apply generally in eukaryotes

Estudio de la reacción dinámica de tubilina en microtubulos. Una simulación qm/am

In this model one-dimensional microtubule is fixed at one of the two and simulated while the opposite end is allowed for growing in random situation. By this study at each step one tubulin has been added to the length for growing microtubule length. Computationally this can be done through generating a uniform random number between (0, 1). Microtubules are demonstrated as straight macromolecules consist of the linear chains of tubulin subunits in the length. QM/MM simulation has been applied to study dynamic instability of the microtubule length. It has been calculated a correct dimension around 10-6 meter of microtubules length consist of around 1650 tubulin dimers.  Microtubule growth rate is related to the soluble tubulin dimer concentration and for all results shown here, simulation of any single condition was run 5–10 times.En este modelo, los microtúbulos unidimensionales se fijan en uno de los dos y se simulan mientras se permite que el extremo opuesto crezca en una situación aleatoria. En este estudio, en cada paso, se ha agregado una tubulina a la longitud para aumentar la longitud de los microtúbulos. Computacionalmente, esto se puede hacer generando un número aleatorio uniforme entre (0, 1). Los microtúbulos se demuestran como macromoléculas rectas que consisten en cadenas lineales de subunidades de tubulina en la longitud. La simulación QM / MM se ha aplicado para estudiar la inestabilidad dinámica de la longitud de los microtúbulos. Se ha calculado una dimensión correcta de alrededor de 10-6 metros de longitud de microtúbulos consiste en alrededor de 1650 dímeros de tubulina. La tasa de crecimiento de los microtúbulos está relacionada con la concentración de dímero de tubulina soluble y para todos los resultados mostrados aquí, la simulación de cualquier condición individual se realizó de 5 a 10 veces

Intraflagellar transport delivers tubulin isotypes to sensory cilium middle and distal segments.

Sensory cilia are assembled and maintained by kinesin-2-dependent intraflagellar transport (IFT). We investigated whether two Caenorhabditis elegans α- and β-tubulin isotypes, identified through mutants that lack their cilium distal segments, are delivered to their assembly sites by IFT. Mutations in conserved residues in both tubulins destabilize distal singlet microtubules. One isotype, TBB-4, assembles into microtubules at the tips of the axoneme core and distal segments, where the microtubule tip tracker EB1 is found, and localizes all along the cilium, whereas the other, TBA-5, concentrates in distal singlets. IFT assays, fluorescence recovery after photobleaching analysis and modelling indicate that the continual transport of sub-stoichiometric numbers of these tubulin subunits by the IFT machinery can maintain sensory cilia at their steady-state length

Differences in intrinsic tubulin dynamic properties contribute to spindle length control in Xenopus species

© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Hirst, W. G., Biswas, A., Mahalingan, K. K., & Reber, S. Differences in intrinsic tubulin dynamic properties contribute to spindle length control in Xenopus species. Current Biology, 30(11), (2020): 2184-2190.e5, doi: 10.1016/j.cub.2020.03.067.The function of cellular organelles relates not only to their molecular composition but also to their size. However, how the size of dynamic mesoscale structures is established and maintained remains poorly understood [1, 2, 3]. Mitotic spindle length, for example, varies several-fold among cell types and among different organisms [4]. Although most studies on spindle size control focus on changes in proteins that regulate microtubule dynamics [5, 6, 7, 8], the contribution of the spindle’s main building block, the αβ-tubulin heterodimer, has yet to be studied. Apart from microtubule-associated proteins and motors, two factors have been shown to contribute to the heterogeneity of microtubule dynamics: tubulin isoform composition [9, 10] and post-translational modifications [11]. In the past, studying the contribution of tubulin and microtubules to spindle assembly has been limited by the fact that physiologically relevant tubulins were not available. Here, we show that tubulins purified from two closely related frogs, Xenopus laevis and Xenopus tropicalis, have surprisingly different microtubule dynamics in vitro. X. laevis microtubules combine very fast growth and infrequent catastrophes. In contrast, X. tropicalis microtubules grow slower and catastrophe more frequently. We show that spindle length and microtubule mass can be controlled by titrating the ratios of the tubulins from the two frog species. Furthermore, we combine our in vitro reconstitution assay and egg extract experiments with computational modeling to show that differences in intrinsic properties of different tubulins contribute to the control of microtubule mass and therefore set steady-state spindle length.This article was prompted by our stay at the Marine Biological Laboratory (MBL), Woods Hole, MA in the summer of 2016 funded by the Princeton-Humboldt Strategic Partnership Grant together with the lab of Sabine Petry (Princeton University). We thank Jeff Woodruff (UT Southwestern), David Drechsel (IMP), and Marcus J. Taylor (MPI IB) for constructive criticism and comments on the manuscript and Helena Jambor for constructive comments on figure design. We thank the AMBIO imaging facility (Charité, Berlin) and Nikon at MBL for imaging support, Aliona Bogdanova and Barbara Borgonovo (MPI CBG) for their help with protein purification, and Francois Nedelec (University of Cambridge) for help with Cytosim. We are grateful to the Görlich lab (MPI BPC), in particular Bastian Hülsmann and Jens Krull, and the NXR for supply with X. tropicalis frogs. We thank Antonina Roll-Mecak (National Institute of Neurological Disorders and Stroke) for help with mass spectrometry analysis and discussions and Duck-Yeon Lee in the Biochemistry Core (National Heart, Lung and Blood Institute) for access to mass spectrometers. For mass spectrometry, we would like to acknowledge the assistance of Benno Kuropka and Chris Weise from the Core Facility BioSupraMol supported by the Deutsche Forschungsgemeinschaft (DFG). We thank all former and current members of the Reber lab for discussion and helpful advice, in particular, Christoph Hentschel and Soma Zsoter for technical assistance and Sebastian Reusch for help with tubulin purification. S.R. acknowledges funding from the IRI Life Sciences (Humboldt-Universität zu Berlin, Excellence Initiative/DFG). W.G.H. was supported by the Alliance Berlin Canberra co-funded by a grant from the Deutsche Forschungsgemeinschaft (DFG) for the International Research Training Group (IRTG) 2290 and the Australian National University. K.K.M. was supported by funds in the Roll-Mecak lab, intramural program of the National Institute of Neurological Disorders and Stroke

Mitosis: Riding the Protofilament Curl

More than 50 years ago, microtubule depolymerization was proposed as the force responsible for chromosome movement. New studies measure the force produced by depolymerization and show that protein ring complexes can couple depolymerization to movement. These results have implications for anaphase chromosome motility and mitotic evolution

Structural and Biochemical Characterization of Cell Shaping Proteins

Microtubules are cytoskeletal filaments in eukaryotic cells where they are required for cell morphogenesis, cell division and intracellular trafficking. Microtubules are highly dynamically assembled from α-/β-tubulin heterodimers. The dynamic instability of microtubules is regulated by several highly conserved microtubule associated proteins (MAPs). In particular, a spatially specialized group of MAPs that accumulate at growing microtubule ends, the plus-end binding proteins (+TIPs), is important to modulate microtubule dynamics in cells. p150glued is one of these +TIPs and is the largest subunit of the dynactin complex. Previous studies of p150glued demonstrated that it functions in recruiting and binding endosomes and dynein to microtubules for initiating retrograde transport. p150glued has two microtubule-binding domains at its N-terminus: a cytoskeleton associated proteins glycine-rich (CAP-Gly) domain, followed by a serine-rich basic domain. To understand how the p150glued CAP-Gly domain and the basic extensions interact with microtubule, cyro-electron microscopic structures of p150glued (1-105)-microtubule complex (CAP-Gly core with its N- terminal basic patch) and p150glued (25-144)-microtubule (CAP-Gly core with its C- terminal basic patch) complex were determined at 9.7 Å and 10.2 Å resolution, respectively. These structures revealed that the CAP-Gly domain binds to the flexible C-terminus of the tubulin (known as E-hook) instead of the core of microtubules. Comparison of the p150glued (1-105)-microtubule reconstruction and p150glued (25- 144)-microtubule reconstruction revealed that CAP-Gly interacts with microtubules very flexibly. In addition, the basic extensions of CAP-Gly core was found to induce microtubule lateral association by neutralization of the negatively charged tubulin C- terminus, which acts as an electrostatic shield to avoid the interaction between individual microtubules. Interestingly, p150glued CAP-Gly together with the basic extensions could induce longitudinal interaction of tubulin for forming curved tubulin oligomers at low temperature, and this process happens in a GTP independent manner. Taken together, p150glued CAP-Gly plus its adjacent basic patches interact with the acidic C-terminus of tubulin and promote tubulin polymerization in two directions, by inducing tubulin longitudinal association at low temperature and lateral interaction once temperature change to physiological condition. Our study about p150glued explained how +TIPs regulate microtubule dynamics from a structure point of view. Cilia are rod-like microtubule based structures protruding from most eukaryotic cells. Cilia are assembled and maintained through a bidirectional transport system called intraflagellar transport (IFT) mediated by IFT complexes and molecular motors moving along axonemal microtubules. The IFT complex is composed of at least 22 polypeptides organized into two complexes named IFT-A and IFT-B. The IFT-B complex is further divided into IFT-B1 and IFT-B2. IFT172, one of the IFT-B2 subunits, is the IFT protein with the highest molecular weight. Chlamydomonas IFT172 is a 1755-amino-acid protein that is encoded by FLA11 gene. The N-terminus of IFT172 contains a WD40 domain, which folds into β-propellers structure while its C-terminus shows tetratricopeptide repeats (TPRs) predicted to form α-helical secondary structure. The domain architecture of IFT172 is highly similar to vesicles coat proteins like COPI and clathrin-adaptor subunits. To characterize IFT172, Chlamydomonas IFT172 was expressed from insect cell and further purified. Surprisingly, IFT172 showed lipid association during the purification and the purification products showed round oligomers containing both IFT172 and membrane. To obtain IFT172 in monomer form instead of the oligomers with lipid, n- Dodecyl β-D-maltoside (DDM) was used and by negative-stain electron microscope observation, the IFT172 monomer was found to adopt two conformations: a globular conformation and a rod-shape conformation. Furthermore, giant unilamellar vesicle (GUV) binding assay was employed to assess the interaction of membrane with IFT172. IFT172 showed high membrane binding affinity and clusters on the membrane surface. To investigate the effect of IFT172 on membrane surface closely, IFT172 with Folch fraction I was investigated under the electron microscope. Vesiculation of 18 nm-diameter small vesicles from the large unilamellar vesicle membrane surface was observed. Further studies revealed that the membrane binding property of IFT172 is mediated by its N-terminal β-propellers, but not C-terminal TPRs. Moreover, I demonstrated that IFT57, the direct binding partner of IFT172 within IFT proteins, competes with membrane for IFT172 binding. These results provided the first evidence that IFT172 binds to membrane through its N-terminal WD40 domains directly and it remodels membrane surface in vitro. Investigation of the functions of IFT172 in vivo is needed to address these issues in the future

Temperature-dependence of microtubule dynamics across Xenopus species

Eukaryontische Zellen besitzen ein Zytoskelett, ein zelluläres Netzwerk aus Biopolymeren. Unter diesen Biopolymeren sind die Mikrotubuli weitgehend konserviert. Diese aus Tubulin aufgebauten Filamente sind dynamisch und wechseln zwischen Phasen des Wachstums und der Schrumpfung. Die genauen Mechanismen, die die dynamische Instabilität der Mikrotubuli bestimmen, werden noch erforscht. Die Allgegenwart von Mikrotubuli wirft die Frage auf, wie sie in verschiedenen thermischen Umgebungen konservierte Funktionen ausführen können. Um dieser Fragestellung nachzugehen, habe ich verwandte Froscharten mit unterschiedlich temperierten Lebensräumen untersucht: Xenopus laevis (16-22 °C), Xenopus borealis (19-23 °C) und Xenopus tropicalis (22-30 °C). Um zu untersuchen, ob sich die biochemischen Eigenschaften von Tubulin und die Dynamik der Mikrotubuli bei den drei Arten an die Temperatur angepasst hat, habe ich die Methoden der Tubulin-Affinitätsreinigung und die temperaturgesteuerte TIRF-Mikroskopie zur Rekonstitution der Mikrotubuli-Dynamik kombiniert. Dabei habe ich festgestellt, dass bei einer Temperatur von 25°C die Wachstumsgeschwindigkeit der Mikrotubuli im Bezug zur thermischen Nische der einzelnen Arten negativ korreliert. Die Verwendung der Arrhenius-Gleichung zum Vergleich der Aktivierungsenergie der Mikrotubuli-Polymerisation für jede Spezies ergab, dass die freie Energie des Tubulins umso höher ist, je kälter die thermische Nische der Spezies ist. Die Mikrotubuli von X. laevis und X. borealis zeigten eine längere Lebensdauer und wurden häufiger zerstört als die von X. tropicalis. Die Tubuline von X. laevis und X. borealis sind phosphoryliert, im Gegensatz zu X. tropicalis. Die Ergebnisse zeigen, dass sich Xenopus Tubulin und die Dynamik der Mikrotubuli an die Temperatur angepasst haben. Kalt lebende Arten kommen mit der niedrigeren Energie des Milieus zurecht, durch verbessertes Wachstum und Stabilität.Eukaryotic cells hold a cytoskeleton, a cellular network of biopolymers. Among the filaments of the cytoskeleton, microtubules are widely conserved. Built from tubulin, those filaments are dynamic, alternating between phases of growth and shrinkage. The biochemical properties of tubulin shape the dynamic behavior of microtubules, which is crucial for many cellular processes. The precise mechanisms determining microtubule dynamic instability are still under investigation. The ubiquity of microtubules raises the question of how they can perform conserved functions within various thermal environments. To address this, I turned to closely related frog species living at different temperatures, Xenopus laevis (niche: 16-22°C), Xenopus borealis (19-23°C) and Xenopus tropicalis (22-30°C). To probe whether the biochemical properties of tubulin and microtubule dynamics adapted to temperature across those three species, I combined tubulin affinity purification and temperature-controlled TIRF microscopy of in vitro reconstitution of microtubule dynamics. I found that at 25°C, the microtubule growth velocity inversely correlates with the thermal niche of each species. Adjusting temperature to each species’ endogenous condition modulates the growth rate differences across species. Using the Arrhenius equation to compare the activation energy of microtubule polymerization for each species suggested that the colder the thermal niche of the species, the higher the free energy of its tubulin. Microtubules from the cold-adapted species X. laevis and X. borealis have longer lifetimes and rescue more often than those of X. tropicalis, both at 25°C and at each species’ endogenous condition. X. laevis and X. borealis tubulins are phosphorylated, contrary to X. tropicalis. My results show that Xenopus tubulin and microtubule dynamics have adapted to temperature. Cold-living species cope with the lower energy of the milieu by facilitating growth and stability

Structural basis for the extended CAP-Gly domains of p150(glued) binding to microtubules and the implication for tubulin dynamics

p150(glued) belongs to a group of proteins accumulating at microtubule plus ends (+TIPs). It plays a key role in initiating retrograde transport by recruiting and tethering endosomes and dynein to microtubules. p150(glued) contains an N-terminal microtubule-binding cytoskeleton-associated protein glycine-rich (CAP-Gly) domain that accelerates tubulin polymerization. Although this copolymerization is well-studied using light microscopic techniques, structural consequences of this interaction are elusive. Here, using electron-microscopic and spectroscopic approaches, we provide a detailed structural view of p150(glued) CAP-Gly binding to microtubules and tubulin. Cryo-EM 3D reconstructions of p150(glued)-CAP-Gly complexed with microtubules revealed the recognition of the microtubule surface, including tubulin C-terminal tails by CAP-Gly. These binding surfaces differ from other retrograde initiation proteins like EB1 or dynein, which could facilitate the simultaneous attachment of all accessory components. Furthermore, the CAP-Gly domain, with its basic extensions, facilitates lateral and longitudinal interactions of tubulin molecules by covering the tubulin acidic tails. This shielding effect of CAP-Gly and its basic extensions may provide a molecular basis of the roles of p150(glued) in microtubule dynamics